Selection system

ABSTRACT

The present invention concerns a method for the selection of a virus comprising the steps of: (a) providing a virus encoding and displaying a fusion polypeptide, said fusion polypeptide comprising a heterologous polypeptide inserted into the sequence of a viral coat protein polypeptide, wherein said virus comprises a cleavable site located within a displayed polypeptide; (b) exposing the virus to a cleaving agent; (c) propagating the virus comprising intact fusion protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 09/710,444 filed on Nov. 10, 2000 which is a USNational Phase Application of PCT/GB99/01523, Publication No. WO99/58655filed on May 13, 1999, which claims priority to Great Britain PatentApplications GB9810223.9 and GB9810228.8 both filed on May 13, 1998. Theentirety of each of the applications is incorporated herein byreference.

The invention relates to a selection system which permits the selectionof polypeptides displayed in a phage display system.

Viruses have been used for the display of peptides and proteins [21, 26,44]. In particular filamentous bacteriophage have been used for displayof proteins and peptides by fusion of the genes encoding the proteins orpeptides to the gene encoding a phage coat protein. As the fusion geneis encapsidated in the phage that is displaying the fusion protein, thisprovides a linkage of phenotype and genotype. Repertoires of proteinscan be encoded by a population of phage, and the rare phage comprisingproteins with predefined binding activities isolated by binding to solidphase. In this way synthetic human antibodies of predefinedantigen-binding specificity have been selected from repertoires ofantibody fragments assembled from different structural elements [10]. Asthe antibody needs to be folded to bind antigen, selection for bindingalso selects for folding. This principle has also been used forselection of folded peptides where binding is mediated by adiscontinuous epitope [8, 11-13].

A problem present in phage display systems is the presence of highlevels of background caused by the presence of phage not displayingdesired polypeptides. For example, antibody repertoires are commonlyencoded as fusion proteins with the p3 protein on phagemid vectors andare encapsidated by the use of helper phage. The helper phage coatprotein competes with the fusion of antibody and coat protein (encodedon the phagemid), leading to phage with “monovalent” rather thanmultivalent display of folded antibody fragments. This can be useful indiscriminating between the affinity and the avidity (with multivalentdisplay) of the antibodies displayed on the phage. However the greatmajority of phages only display the helper phage coat protein whichcontributes to a “background” binding to antigen. In this case it isdesirable to select for phages that display folded antibodies, and toeliminate those that do not.

Moreover, all of the systems in current use rely on a binding activityin the polypeptide to be selected in order to perform the isolation ofthe desired display bodies from those which do not encode polypeptideshaving a desired characteristic. This places a limitation on availabledisplay systems to the selection of folded polypeptides which possess aknown binding activity. It would be desirable to have a means forselection of displayed proteins or polypeptides that is independent ofthe binding activity thereof.

For example, there is considerable interest in building folded proteinsde novo. Attempts have been made to design proteins de novo by assemblyof predefined elements of secondary structure and also from randomsequences (for review [5]). In some cases the designed proteins havebeen shown to retain elements of secondary structure but lack the stabletertiary interactions characteristic of the folding of native proteins,suggesting the presence of molten globules (see [6] and referencestherein). More successful has been the creation of native-like proteinsbased on a pre-existing backbone [7,8]. In these cases the bindingactivities of a de novo designed protein will be unknown. In this caseit is desirable to select for phages displaying folded proteins, and toeliminate those that do not.

Although attempts have been made to screen for folded proteins by theirability to survive degrading enzymes in bacteria [16-18], such methodsdo not allow for selection if bacterial growth or survival does notdepend on the function of the folded protein. Thus, these systems areonly applicable to a small minority of polypeptides which one might wishto select according to the ability to fold.

It has previously been shown that the insertion of a peptide sequencebetween a proteolytically stable tag fused to the minor phage coatprotein p3 and the p3 protein itself, followed by proteolysis, providesa means to select for phages bearing peptide sequences that aresusceptible to proteolysis [19, U.S. Pat. No. 5,780,279]. In theseexperiments, phage are bound to an affinity resin binding an N-terminal,proteolytically stable tag on the phage. If the bound phage aresubjected to proteolysis and elution, only phage with cleavablesequences are eluted. This method is used to identify, among arepertoire displayed on phage, amino acid sequences suitable assubstrates for proteases. The sequences introduced are short and wouldnot be capable of folding independently. Moreover, the system selectsspecifically for eluted rather than bound phage; in other words, it isspecifically configured to isolate cleaved rather than uncleaved phage.

SUMMARY OF THE INVENTION

The present invention exploits the application of peptide cleavage toeliminate unwanted viruses.

According to a first aspect, therefore, the invention provides a methodfor the selection of a virus comprising the steps of:

(a) providing a virus encoding and displaying a fusion polypeptide, saidfusion polypeptide comprising a heterologous polypeptide inserted intothe sequence of a viral coat protein polypeptide, wherein said viruscomprises a cleavable site located within a displayed polypeptide;(b) exposing the virus to a cleaving agent;(c) propagating the virus comprising intact fusion protein.

According to the present invention, virus may be selected by cleavage ofnon-resistant virions using a cleaving agent. As used herein, “virus”refers to an infective inocculum of virions, which may incorporatecleavage sites, optionally as part of heterologous polypeptides encodedby the viral genome. Thus, “virus” may refer to a plurality of virions,such that it may encode a repertoire of polypeptides; alternatively, asthe context requires, it may be used to denote a single virion. The term“virus” includes any suitable virus which may incorporate a cleavagesite, either naturally or through manipulation. A preferred virus foruse in the present invention is bacteriophage, preferably filamentousbacteriophage.

The term “polypeptide” is used generally to denote molecules constructedof a plurality of amino acids, the amino acids being joined togethercovalently such as through peptide bonds. “Fusion” polypeptides areessentially polypeptides which are incorporated into viral coatproteins, such that a fusion is created between the viral coat proteinand the polypeptide in question. The fusion may incorporate thepolypeptide into the viral coat protein, advantageously between domainsthereof, or place it at one end thereof, to make a terminal fusion. Thepolypeptide is referred to as a “heterologous” polypeptide, to denotethat it is heterologous to the viral coat protein into which it isinserted. It is possible, however, that it is derived from anotherpolypeptide of said virus.

In one sense, polypeptide is used interchangeably with “protein” herein,in that no difference of structure or size is implied. Substantially anypolypeptide may be selected for by the method of the present invention,including structural polypeptides, polypeptides having enzymaticactivity and polypeptides having binding activity, including antibodiesand antibody fragments. Cleavage sites may be present in thepolypeptides, and may be naturally-occurring or may be engineered intothe polypeptide or into a linker peptide attached thereto. “Polypeptide”may also refer to inserted polypeptides which are essentiallynon-folding polypeptides and serve to encode a cleavable site and insertthis site into the coat protein of a virus. Inserted polypeptides maytake the form of N- or C-terminal fusions, or may form part of the coatprotein itself.

A “cleavable site” is a site capable of cleavage when exposed to acleaving agent. In the present invention, the use of protease cleavagesites, capable of being cleaved with proteases, is preferred. Proteasecleavage sites may be part of, or incorporated in, polypeptidesaccording to the invention; alternatively, it may be independentlyengineered into the coat protein of the virus. A feature of thecleavable site is that it should either be absent from the virus otherthan at the site of its specific insertion according to the presentinvention, or otherwise inaccessible to cleavage, or present only inviral proteins which are not required after virion assembly to mediateinfection.

In accordance with the invention, the cleavable site may be insertedinto or present in any suitable position in the virus. Advantageously,however, it is inserted into or present in either the coat proteinitself or the heterologous polypeptide which forms part of the fusionpolypeptide.

In a preferred aspect of the present invention, more than one cleavablesite may be used. For example, one site may be inserted or otherwiseknown to be present in the virus, whereas the presence of another sitemay be unknown or dependent on randomisation of the heterologouspolypeptide sequence. In a particularly preferred embodiment, thecleavable sites may comprise a protease cleavable site and a bond formedthrough sidechains on one or more amino acids, such as a disulphidebond. Disulphide bonds are cleavable by reducing agents, such as DTT orβ-mercaptoethanol.

If a virus contains a disulphide bond, cleavage of a protease cleavablesite located between the two cysteine residues which form the bondthrough their sidechains will not lead to loss of viral infectivitysince the disulphide bond is capable of retaining the covalent linkageof the viral polypeptides.

Thus, the invention further provides a method for identifying thepresence of disulphide bonds in polypeptides.

Conversely, in the event that the selection of disulphide-containingpolypeptides is not desired, viruses are advantageously treated with areducing agent before or after proteolysis, in order to eliminatebackground due to viruses which have been cleaved by the protease butwhich have been held together by disulphides.

The fusion polypeptide may comprise one or more heterologouspolypeptides. In the case of a terminal fusion, one such heterologouspolypeptide may function as a protein tag, allowing phage which expressthe fusion polypeptide to be identified. The cleavable site may, in sucha case, be positioned in or near the tag, such that cleavage of thecleavable site releases the tag.

A tag is any suitable entity capable of binding to a ligand which may beused to isolate a virus by the method of the present invention.Accordingly, the tag is resistant to the cleaving agent used in themethod of the invention. Examples of tag/ligand pairs includebarnase/barstar, avidin/biotin, antibody or antibody fragments andligands, chelating groups and chelates, for example metals, and thelike.

In all embodiments of the present invention, which are described ingreater detail below, the uncleaved polypeptides are selected for; thecleaved material is discarded in the selection step.

Preferably, the virus according to the invention encode a repertoire ofheterologous polypeptides. A repertoire is a collection of members,preferably polypeptides, which differ slightly from each other in arandom or partially randomised manner. Preferably, a repertoire ofpolypeptides is a collection of variant polypeptides which preferablyincorporate random or partially randomised mutations. As used herein, arepertoire preferably consist of 10⁴ members of more. A repertoireadvantageously comprises a very large number of members, typicallybetween 10⁸ and 10¹¹, and potentially 10¹⁴ or higher.

The heterologous polypeptide, or repertoire of such polypeptides, isadvantageously displayed on the surface of the virus which encodes it,by virtue of its being incorporated into a coat protein, or on thesurface of cells infected by the virus. Where the virus is abacteriophage, the protein may also be displayed on the surface ofbacteria infected with the bacteriophage.

The cleavable site is advantageously located in or adjacent to theheterologous polypeptide, such that it can be protected by folding ofthe heterologous polypeptide and thus allow selection for heterologouspolypeptides which are capable of correct folding. Alternatively,however, the cleavable site may be located distal to the heterologouspolypeptide; in such embodiments, the cleavable site may serve to permitreduction of background in phage display techniques. For example,introduction of the cleavable site into helper phage used with phagemidencoding a repertoire of polypeptides allows helper phage to be removedby cleavage prior to infection of host cells, thus dramatically reducingbackground due to “empty” phage. Advantageously, therefore, thecleavable site is incorporated into the virus coat protein.

As referred to herein, a phagemid is a plasmid cloning vector whichcomprises viral replication sequences but is deficient in at least oneviral function. This means that whilst phagemid may be inserted intohost cells by conventional nucleic acid transfer methods, and will existin the host cells in an episomal state, they are unable to assemble intovirions and thus complete a viral cycle of infection. Helper phage areused to supply the deficient viral functions and permit the phagemid tobe packaged into virions. In accordance with the invention, phagemid mayencode coat protein fusions with heterologous polypeptides whichincorporate a cleavable site.

Helper phage provide the viral function lacking in phagemid in order toallow packaging of the phagemid into virions. According to theinvention, helper phage may be modified in order to render themcleavable by a cleaving agent, for example a protease. In one aspect ofthe present invention, helper phage may incorporate a coat proteinhaving a cleavable site which, when cleaved in the “rescued” progenyphage, will render the helper phage-derived coat protein unable tomediate infection.

As will be apparent from the forgoing, in the method according to thepresent invention the virus which are resistant to cleavage areselected. Advantageously, the resistant virions will be selected byinfection of susceptible host cells, such as bacterial host cells.Cleaved virions are not infective. Alternatively, binding of virions toa ligand, for example via a tag, is dependent on protection of acleavable site in the virus, such that viruses which are cleaved are notisolated by ligand/tag binding.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be configured in a number of ways according tothe intended procedure to which it is desired to apply the basicmethodology. Selective cleavage of virions is used to reduce backgroundin phage display techniques. By cleaving and removing phage which eitherdo not contain a heterologous polypeptide, or express a heterologouspolypeptide which is not capable of correct folding, the sensitivity ofa phage display process can be increased substantially. As demonstratedin the experimental section below, using the methodology of theinvention phage display may be used to select polypeptides which are notsusceptible to selection by display techniques according to prior artmethods.

In a first configuration, cleavage of a cleavable site in a virion coatprotein may be exploited to reduce background attributable to phagedisplaying no heterologous polypeptides, or phage displayingheterologous polypeptides which are incapable of folding correctly.Cleavage of displayed polypeptides, in accordance with the presentinvention, results in the impairment of the viruses to achieve infectionof host cells. Thus, by propagating viruses which have been exposed to acleavage agent, it is possible to enrich the virus for virions whichcomprise displayed polypeptides which are resistant to cleavage. As usedherein, “impair” means to reduce; it thus includes but partial andcomplete prevention of infection of host cells by affected virus.

The coat protein is selected as the site for cleavage on the groundsthat it is available to cleaving agents in the host cells harbouring thevirus or at the surface of the virus itself. Thus, in a preferredembodiment, virus preparations may be treated with a cleaving agent, inorder to render virions having cleavable coat proteins unable to mediateinfection of host cells. Alternatively, cells infected with the virusmay be treated with a cleaving agent active within the cell, which willprevent packaging of virus comprising a cleavable coat protein. As usedherein, the terms pIII, p3, g3 and gene III are equivalent alternativeabbreviations for bacteriophage coat protein III.

According to the present invention, reference to selection may beinterpreted as a reference to screening, since the same processes may beused to screen phage, as will be apparent to persons skilled in the art.

Cleavable sites may be naturally part of the coat protein, butpreferably they are engineered therein. Preferred cleavable sitesinclude protease cleavage sites, which may be found in polypeptides orengineered as an integral part of their sequence. Typically, proteasecleavage sites may be defined in terms of amino acid sequences which aresusceptible to cleavage by a protease. For example, the inventionencompasses the use of protease cleavage sites cleavable by one or moreof the proteases trypsin (cleaves at Lys, Arg), chymotrypsin (Phe, Trp,Tyr, Leu), thermolysin (small aliphatic residues), subtilisin (smallaliphatic residues), Glu-C (Glu), Factor Xa (Ile/Leu-Glu-Gly-Arg), Arg-C(Arg) and thrombin.

Protease cleavage sites may be incorporated into the coat protein of avirus by constructing a fusion between the coat protein and a furtherpolypeptide, the further polypeptide containing the cleavage site. Thefurther polypeptide should be inserted at a position in the viral coatprotein such that it allows the assembly of a functional viral capsidand subsequent infection, but if cleaved will result in the impairmentof infectivity.

If the protease cleavage site incorporated in the coat protein remainsuncleaved, therefore, the virus is capable of assembly into functionalvirions and retains the potential to infect host cells. If the proteasecleavage site is cleaved, however, the structure of the viral coatprotein will be compromised and the virus will lose at least part of itspotential to infect host cells.

In a preferred embodiment, the virus for use in the present invention isa bacteriophage, preferably filamentous bacteriophage. Filamentousbacteriophage is widely used in phage display techniques for theselection of polypeptides from phage libraries encoding a largerepertoire thereof. Conventionally, the repertoire of polypeptides isinserted in the p3 protein of filamentous bacteriophage, but any othersuitable site may be employed within the scope of the present invention.

In the case of the p3 protein of filamentous bacteriophage the proteinconsists of three domains. The N-terminal D1 is involved in binding tothe to 1A receptor, D2 in binding to the F-pilus (and mediatinginfection) and D3 in anchoring the protein to the phage particle.Peptides and proteins can be inserted at the domain boundaries withoutabolishing infectivity [21, 22], but the presence of all the domains isessential for phage infectivity [23]. The bacteriophage are resistant toproteolysis (allowing their use as “substrate” phage, [19]), but theintroduction of polypeptides comprising protease cleavage sites into p3,for example at the junctions between domains leads to loss ininfectivity of the phage upon proteolysis.

The protease cleavage sites may be incorporated into heterologouspolypeptides. As described above, heterologous polypeptides may beencoded in the form of a repertoire in a phage library. As foldedpolypeptides or proteins are often resistant to proteolysis and unfoldedproteins are sensitive, cleavage requires the polypeptide chain to bindand adapt to the specific stereochemistry of the protease active site,and therefore to be flexible, accessible and capable of local unfolding[14, 15]. The cloning of a polypeptide comprising protease cleavagesites at the domain junctions of p3, followed by proteolysis, provides ameans of selection for phages bearing proteins that are resistant toproteolysis and are folded.

In the case of phage display repertoires (wherein the polypeptide to beselected is cloned at the N-terminus of p3) encoded on phagemid vectors,the use of helper phage comprising a polypeptide comprising proteasecleavage sites at the domain boundaries, followed by proteolysis,provides a means of selection for phages that display the fusion proteinby eliminating the helper phage after the “help” has been given.

The use of the protease-cleavable helper followed by protease cleavageselects for phages bearing the fusion protein (and for good display). Asmany phages in a repertoire do not display fusion proteins [26] andthese contribute to non-specific binding of the phage, this should alsoimprove selection efficiencies. When using the techniques of the priorart, only between 0.1-1% of all phage particles in a phage library maycomprise a gene 3 protein arising from the phagemid. Therefore themajority (99-99.9%) of phage particles that have bound non-specificallyto the solid support used in selection will comprise p3 from the helperphage (irrespective of the genome carried by the phage particle whichmost likely will be a phagemid DNA), these particles are renderednon-infective by proteolytic cleavage.

According to a third embodiment, the selection process may be used foridentification of interacting protein elements. If two such elementslinked by a polypeptide comprising protease cleavage sites are clonedbetween the D2 and D3 domains for display on phage, the only infectiousphages after proteolysis are those in which the D2 and D3 domains areheld together by non-covalent interactions between the interactingprotein elements. The invention accordingly permits selection of arepertoire of polypeptides for its ability to interact with a selectedpolypeptide, or a second repertoire of polypeptides. Unlike thetwo-hybrid system, the invention relies on dissociation ofnon-interacting elements as distinct from the association of interactingelements for the selection step. Moreover, the invention permits theharnessing of the power of phage display to greatly increase the degreeof selection.

The invention optionally comprises the use of conditions or agents,during cleavage of the cleavable site, which modulate the liability ofthe cleavage site in the presence of the cleaving agent. This approachmay be used to increase cleavage, for example to select only forpolypeptides which fold in such a manner as to closely shield thecleavable site from access by the cleaving agent, or to decreasecleavage, for example to select stable mutants from a repertoire ofpolypeptides which is ordinarily relatively labile under cleavageconditions.

For example, modulation of the liability of the cleavable site may beachieved by the use of agents which increase or decrease such liability.Thus, a protein denaturant may be included, at a suitable concentration,to destabilise a polypeptide and render it more labile. Alternatively, aligand for a polypeptide may be included. The ligand may stabilise thefolded structure of the polypeptide, rendering it less sensitive tocleavage. Alternatively the ligand may destabilise the folded structureof the polypeptide, for example by favouring the adoption of analternative configuration. This may render the polypeptide moreaccessible to the cleavage agent, and thus more labile.

In a further embodiment, the conditions of the cleavage process may bealtered, such as by manipulating the pH or the temperature at whichcleavage is conducted, to achieve similar effects. Thus, deviation ofthe pH from the optimum for the polypeptide comprising the cleavablesite may cause the site to become more accessible to the cleaving agent.Similarly, raising (or lowering) the temperature of the conditions underwhich the polypeptide is cleaved may render the polypeptide more or lesssusceptible to cleavage.

In some instances, non-covalent interactions may be responsible forpeptides retaining their structure and coat proteins remaining viable,even after successful cleavage of the cleavable site. The use ofdenaturants, temperature variation and other potentially destabilisingtechniques may also be used to decrease the likelihood of a cleavedpolypeptide retaining its structure.

Proteolytic selection for protein folding may be applied in a number ofareas, as it allows much larger numbers of proteins to be processed thanwith conventional screening. For example, it allows the isolation ofmutant proteins with improved stability [1], for example fromcombinatorial libraries of mutants in which residues at several sitesare varied simultaneously [39, 40] or from random mutants or byrecombination [3, 4]. It also allows the isolation of novel proteins andarchitectures from large repertoires of sequences [16-18, 41]; and forimprovement in folding stability over several rounds of mutation andincreasingly stringent selection, much like the affinity maturation ofantibodies.

A second configuration of the present invention concerns the use of tagsto allow isolation of correctly folded heterologous polypeptides,exploiting the ability of correctly folded polypeptides to protect acleavable site on to or near to an associated tag. The insertion of apolypeptide between the stable tag fused to the N-terminus of the viralcoat protein and the coat protein itself, followed by cleavage, providesa means of selection for virus bearing proteins that are resistant toproteolysis and are folded. Thus only virions, whose insertedpolypeptide is not degraded, will keep the tag fusion as part of theircoat, and only these virions can therefore be captured by affinitypurification using this tag. After elution the affinity captured phasesfrom the ligand, these phages can be propagated and subjected to furtherrounds of the same selection procedure.

Alternatively, virions may be bound to an affinity matrix, comprising aligand for the tag, prior to cleavage. The cleaving agent maysubsequently be added, and only resistant phage will be retained on thematrix. These may then be eluted as required.

Suitable matrices include columns, beads and other surfaces to which aligand for the tag is bound.

According to the present invention, reference to selection may beinterpreted as a reference to screening, since the same processes may beused to screen phage, as will be apparent to persons skilled in the art.

Cleavable sites are substantially as described for the previousconfiguration of the present invention and are advantageously proteasecleavable sites.

Cleavage requires the polypeptide chain to bind and adapt to thespecific stereochemistry of the protease active site, and therefore tobe flexible, accessible and capable of local unfolding [14, 15]. Foldedpolypeptides or proteins are often resistant to proteolysis, due to arelative inflexibility in their structure, whilst unfolded proteinsremain sensitive.

As referred to above, the possible selection of polypeptides from arepertoire which, through variation or mutation, do not contain arecognition sequence for any particular protease used in this method,can be circumvented in two ways. For example, the use of a cocktail ofproteases with very distinct recognition sequences would ensure that allpolypeptides should be cleavable, if not protected by their foldedstatus. Alternatively, a phage repertoire of polypeptides to be selectedcould be partially denatured, such that the inserted polypeptide unfoldsbut the phage and the N-terminal tag remains intact. Proteolyticdigestion followed by affinity purification would remove all phages fromthe repertoire, which have escaped proteolysis due to the lack ofprotease recognition sequences in the polypeptide. Phages not bound bythe resin, contain only phages, which contain the protease recognitionsequence in the polypeptide displayed and which may or may not escapeproteolysis under non-denaturing conditions. Thus these would besubjected to proteolytic selection based on protection by the foldingstatus of the polypeptide displayed.

The selection process may also be used for the identification ofinteracting protein elements. Thus if two such elements linked by apolypeptide comprising protease cleavage sites are cloned between theN-terminal, proteolytically stable tag for display on phage and the coatprotein, the only phages after proteolysis, that can be captured viaaffinity binding to the tag, are those in which the tag and the p3protein are held together by non-covalent interactions between theinteracting protein elements.

The invention is further described in the following examples, for thepurposes of illustration only.

EXAMPLE 1 Resistance of Filamentous Phage to Proteolysis

Materials and Methods for Examples 1-6 are appended at the end ofExample 6.

Phage is incubated under a range of denaturing conditions in vitro andthen restored to native conditions immediately before infection ofbacteria. The incubation of phage in 10 M urea, or extremes of pH (aslow as pH 2, and as high as pH 12) and temperature (as high as 60° C.)did not lead to a major loss of infectivity (Table 1). This indicatesthat the phage is either resistant to denaturing conditions or that ifit does unfold it is able to refold rapidly. However with guanidinehydrochloride (GndHCl) a 5 fold loss in phage infectivity is observedabove 5 M and a further 5 fold loss at 8 M (Table 1).

Phage is then incubated under native conditions with a range ofproteases (trypsin, Factor Xa, IgA protease, Asp-N, chymotrypsin, Arg-C,Glu-C, thrombin, thermolysin, subtilisin) with different specificities.There is no loss in infectivity except for subtilisin which has beenreported to cleave the p3 protein [24]. If phage is incubated underdenaturing conditions in the presence of proteases such as trypsin in3.5 M urea (or >47° C.), infectivity is lost. This indicates that underdenaturing conditions the unfolding of the phage coat proteins issufficient to make sites available for proteolysis.

EXAMPLE 2 Construction of Phage with Protease Cleavage Sites

A sequence (PAGLSEGSTIEGRGAHE, SEQ ID NO: 1) comprising severalproteolytic sites is inserted in the flexible glycine-rich regionbetween the D2 and D3 domains of the phage p3.

Incubation of the phage (fd-K108) under native conditions with trypsin,thermolysin or subtilisin now resulted in almost complete loss ofinfectivity (from 10⁷ to <10 TU/ml) and incubation with Glu-C andchymotrypsin resulted in a major loss (from 10⁷ to 10⁴ TU/ml). Thisindicates that these proteases cleave the new linker. However incubationwith Factor Xa, Arg-C or thrombin did not lead to a loss in infectivity,despite the presence of potential cleavage sites for these enzymes.Presumably the presence of the D2 and D3 domains may block access orcleavage for these enzymes in the case of the present polypeptide.

EXAMPLE 3 Construction of Protease Cleavable Helper Phage and Phagemid

Fusion of proteins to p3 should lead to multivalent display of theprotein on the phage. However if the protein is fused to p3 encoded by aphagemid (such as pHEN1 [25]), and the bacteria harbouring the phagemidis rescued with a helper phage (such as VCSM13), the fusion protein hasto compete for incorporation into the phage with the helper p3. Thisleads to so-called “monomeric” phage, in which usually less than onecopy of the fusion protein is attached to each phage particle [26].

The use of “monomeric” phage might be expected to be advantageous forselection of high affinity interactions. Furthermore fusion proteins in“monomeric” phage should be more sensitive to proteolysis, asinteractions between multimers of fusion protein are avoided. However adisadvantage is that the majority of infective phages do not display aprotein; such phages binding non-specifically to solid phase areamplified during each round of phage growth.

Protease cleavable helper phage are therefore constructed, byintroducing the protease cleavage sequence between the D2 and D3 domainsto generate the helper phage KM13.

TABLE 1 Stability of wild type fd-DOG under different conditions. Urea(60° C., 90 min) Control 2 M 4 M 6M 8 M 10 M 0.56 0.64 0.32 0.32 0.80068 GndHCl (37° C., 90 min) Control 2 M 4 M 5 M 6 M 7 M 8 M 0.72 0.600.70 0.16 0.13 0.16 0.03 pH (37°, 30 min) Control pH 2.2 pH 4.0 pH 7.4pH 10 pH 12 1.5 0.46 1.3 1.5 1.4 0.40 Temperature (30 min) Control 22°C. 37° C. 60° C. 9.7 8.3 9.6 12.0 The infectivity (TU/ml × 10¹⁰) wasmeasured (see Materials and Methods) and has an estimated error of about±6%.

KM13 is shown to rescue the phagemid pHEN1. Furthermore trypsin is shownto cleave a major fraction (about 50%) of p3 of the rescued phage asshown by Western blot and detected with an anti-D3 mAb (FIG. 1). Howeverphage infectivity is hardly altered by the cleavage; it thereforeappears that only a fraction of the p3 need be entire to mediatebacterial infection.

KM13 is also shown to rescue a pHEN1 phagemid encoding a single chainantibody fragment [27]. Here cleavage by trypsin resulted in a 50 foldloss in phage infectivity (data not shown), consistent with indicationsthat only a small fraction of the phage express fusion protein whenrescued with helper phage [26, 28].

A protease cleavable phagemid is also constructed. The phagemid can berescued with KM13 or VCSM13. As expected, infectivity of this phagemidrescued with KM13 (but not VCSM13) is destroyed by trypsin. Thisphagemid vector is prone to deletions in the D2-D3 linker; by changingthe codon usage in the linker regions on either site of the proteasecleavable site, and shortening the length of these linker regions, amore stable vector is created (pK1; FIG. 2). In a second vector (pK2;FIG. 2), the sequence of the polylinker is arranged so as to place D3out of frame to render religations within the polylinker non-infectious.

EXAMPLE 4 Construction of a Phage Antibody Library Using ProteaseCleavable Helper Phage

Bacteria are electroporated with phagemid DNA encoding a repertoire ofscFv fragments fused to the N-terminus of p3 and grown in liquid culture(2×TY containing antibiotic to select for bacteria containing phagemidand glucose to suppress expression of gene 3). In the mid log phase ofbacterial growth (OD600=0.5) the helper phage KM13 is added to thebacteria to give a ratio of helper phage to bacteria of 20:1. Thebacteria is incubated at 37° C. without shaking for 45 min then withshaking for 45 min. The bacteria are harvested by centrifugation andresuspended in fresh medium containing 50μ/ml kanamycin and antibiotic,without glucose, to select for presence of phagemid DNA. The culture isgrown overnight at 30° C. with shaking.

Bacteria are removed from the phage containing supernatant bycentrifugation. Phage is precipitated from the supernatant by adding ⅕the volume of 20% PEG/2.5 M NaCl. After 1-2 hours at 4° C. theprecipitated phage is collected by centrifugation. The phage isresuspended in PBS (a second PEG precipitation is optional) and can beused in selection.

The library of phages is allowed to bind to antigen (immobilised onsolid support such as an immunotube or in solution to tagged (i.e.biotinylated) antigen which can be immobilised after affinity binding ofphage antibodies). Unbound phage is removed by extensive washing (thestringency of washing can be varied with respect to time and detergentsadded).

Phage libraries comprising a cleavable tag, such as the c-myc taginserted between the antibody and gene 3, can be eluted by addition oftrypsin in solution at a concentration of 0.1 to 1 mg/ml. (Phagelibraries without a cleavable sequence between the antibody and gene 3can be eluted by adding 100 mM Triethylamine. In this case the solutionis neutralised by adding 1 M Tris-HCl pH 7.4. and after 10 min, trypsinadded to a final concentration of 0.1 to 1 mg/ml.) Trypsin also cleavesthe copies of gene 3 from the helper phage, while leaving gene 3 fromthe phagemid intact. Thus only phage that had carried, or still carry,an antibody fusion will be infective.

Phage is used to infect bacteria in mid log phase of growth (OD600=0.5),and the bacteria is plated on agar plates containing antibioticselecting for phagemid DNA. Individual clones were picked and phageprepared as above. The resulting phage is used in ELISA to identifyphage antibodies binding specifically to the antigen of interest.

EXAMPLE 5 Selection for Folding Using Barnase as a Model

Barnase is a small RNase of 110 amino acid residues whose folding hasbeen extensively studied (for review [2]). Barnase contains multiplesites for trypsin cleavage, although the folded protein is resistant tocleavage (data not shown). Phage with barnase cloned between D2 and D3should therefore be resistant to protease cleavage and capable ofselection.

As barnase is toxic to Escherichia coli, a mutant A (His 102->Ala) iscloned which is catalytically inactive but stable [29, 30] into thephagemid pK2. A mutant B (His102->Ala,Leu14->Ala) is also cloned, withlower stability; Leu14 is buried in the hydrophobic core and itsmutation creates a large cavity in the core affecting the packing ofdifferent structural elements [31]. The phages (rescued with KM13) bindto the inhibitor barstar by ELISA (FIG. 3), and therefore display themutant barnase in a folded form.

The phages are then incubated with trypsin at a range of temperatures(FIG. 4). After incubation at 10° C., there is a decrease in phageinfectivity of 5 to 10 fold for both mutants, suggesting that (as abovewith the display of scFv fragment) only a small fraction of the phagesdisplay the fusion protein. There is no further loss in infectivity oncleavage until 30° C. (for mutant B) or 37° C. (for mutant A). In bothcases the major transition is at least 10° C. below that expected forthe reversible thermal unfolding of the mutants.

Phages A and B are mixed in different ratios and incubated with trypsinat 20° C., where both mutants are stable to cleavage, or at 37° C. whereonly A is stable. After “proteolytic selection” the phages are platedand analysed by PCR, which is followed by restriction digest todistinguish the mutants. As shown in the Table 2, mutant A is enrichedby a factor of 1.6×10⁴ after a single round and by 1.3×10⁶ after tworounds of proteolytic selection at 37° C. No enrichment can be detectedat 20° C.

EXAMPLE 6 Selection for Folding Using Villin as a Model

The 35 amino acid subdomain of the headpiece domain of thef-actin-bundling protein villin [32] is much smaller than barnase. Itnevertheless forms a stable fold at room temperature and is resistant toproteolysis; furthermore its stability does not rely on disulphide bondsor binding ligands [33]. The villin subdomain (which contains severalpotential trypsin cleavage sites) is cloned between the D2 and D3domains of the phage, and incubated with trypsin at differenttemperatures. The profile for loss of infectivity is not as sharp aswith barnase, with the major transition below 35° C., considerably belowthe thermal unfolding of villin (70° C.) [32, 33]. The phage displayingvillin are mixed with phage, which were produced using the phagemid pK1and the helperphage KM13, and incubated with trypsin. After a singleround of proteolytic selection, the villin fusion phage are enriched8.7×10³ fold (Table 3).

In summary, the results from Examples 1 and 2 show that the infectivityof the phage is relatively resistant to temperature, pH, urea andGndHCl, and to several proteases, but if a flexible linker comprising aprotease cleavage site is inserted between domains D2 and D3 of thephage coat protein p3, the phage becomes sensitive to cleavage. Bycontrast, as shown in Examples 5 and 6, if the protease cleavage sitescomprise a folded protein domain such as barnase or villin, the phage isresistant to cleavage. This allows proteolytic selection for proteinfolding, with enrichment factors of greater than 10⁴ fold for a singleround of selection. Selection is evident for both for barnase, anaverage sized [34] domain of 110 amino acids and for villin, a smalldomain of 35 amino acids.

Discrimination between structures of different stabilities can beaccomplished by increasing the stringency of proteolytic selection. Thuswith increase in temperature, both barnase and villin became susceptibleto cleavage, reflecting protein unfolding. However the main impact ofprotease cleavage is at a temperature lower than the unfoldingtransition as measured by circular dichroism [38]. This may reflect thefact that the unfolding transition is a fully reversible process,whereas the cleavage by proteases (of unfolded structure) is a kineticand irreversible process, pulling over the equilibrium from folded tounfolded (and cleaved) structure. This is consistent with the CDunfolding transition seen with villin [33], where at temperatures as lowas 35° C. there is evidence of unfolding, the same point at which villinstarts to become susceptible to protease attack.

TABLE 2 Selection of Barnase mutants. Phage A:Phage B 1.1^(a) 1:10²1:10⁴ 1:10⁶ 1:108 Round 1 Phage A 16 (14^(b)) 24 20 0 nd Phage B  8(10^(b)) 0 4 24 nd Enrichment — — 1.6 × 10⁴ — nd Round 2 Phage A nd ndnd 24 nd Phage B nd nd nd 12 36 Enrichment nd nd nd 1.3 × 10⁶ — Mixturesof barnase mutants (A + B) in ratios from 1:1 to 1:10⁸ were selected byproteolysis at 37° C., 24 (or 36 in round 2) phage clones analysed andnumbers of each mutant noted above. ^(a)Selection at 20° C. where bothmutants are expected to be stable, ^(b)before selection.

TABLE 3 Selection of villin. 1:1 1:10² 1:10⁴ 1:10⁶ pK1  0 (16^(a)) 0 724 Villin 24 (8^(a)) 24 17 0 Enrichment — — 8.7 × 10³ — Mixtures ofvillin-phage and pK1 rescued with KM13 in ratios from 1:1 to 1:10⁶ wereselected by proteolysis at 10° C., 24 phage clones analysed and numberof each noted above. ^(a)before selection.

TABLE 4 Primer sequences Pklinker 5′ GGCACCCTCAGAACGGTACCCCACCCTCAGAGGCCGGCTGGGCCGCCACCCTCAGAG 3′ (SEQ ID NO: 2) po1yXafor5′ GGTGGCGGCCCAGCCGGCCTTTCTGAGGGGTCGACTATAGAAGGACGAGGGCCCAGCGAAGGAGGTGGG GTACCCCCTTCTGAGGGTGG 3′ (SEQ ID NO:3) po1yXaback 5′ CCACCCTCAGAAGGGGGTACCCCACCTCCTTCGCTGGGCCCTCGTCCTTCTATAGTCGACCCCTCAGAA AGGCCGGCTGGGCCGCCACC 3′ (SEQ ID NO:4) fdPCRBack 5′ GCGATGGTTGTTGTCATTGTCGGC 3′ (SEQ ID NO: 5) LIBSEQfor5′ AAAAGAAACGCAAAGACACCACGG 3′ (SEQ ID NO: 6) LIBSEQback5′ CCTCCTGAGTACGGTGATACACC 3′ (SEQ ID NO: 7) LSPAfor5′ GTAAATTCAGAGACTGCGCTTTCC 3′ (SEQ ID NO: 8) LSPAback5′ ATTTTCGGTCATAGCCCCCTTATTAG 3′ (SEQ ID NO: 9) Flagprimer5′ CAAACGGGCGGCCGCAGACTACAAGGATGACGA CGACAAGGAAACTGTTGAAAGTTGTTTAGCAA 3′(SEQ ID NO: 10) RECGLYfor 5′ CCCCTCAGAAAGGCCGGCTGGGCCGCCGCCAGCATTGACAGGAGGTTCAGG 3′ (SEQ ID NO: 11) RECGLYback5′ GAAGGAGGTGGGGTACCCGGTTCCGAGGGTGGT TCCGGTTCCGGTGATTTTG 3′ (SEQ ID NO:12) delCKpn 5′ CCCTCGGAACCGGTACCCCAGCTGCTTCGTGGG CCC 3′ (SEQ ID NO: 13)Barnasefor 5′ CTGGCGGCGGCCCAGCCGGCCCTGCACAGGTTA TCAACACGTTTGAC 3′ (SEQID NO: 14) BarnaseH102Aba 5′ CTCGGAACCGGTACCTCTGATTTTTGTAAAGGTCTGATAAGCG 3′ (SEQ ID NO: 15) ck villinfor5′ GGCGGCCCAGCCGGCCTTTCTCTCTCTGACGAG GACTTCAAGGC 3′ (SEQ ID NO: 16)villinback 5′ CCTCGGAACCGGTACCGAAGAGTCCTTTCTCCT TCTTGAGG 3′ (SEQ ID NO:17)

Materials & Methods (Examples 1-6) Materials

All restriction enzymes, T4 ligase are obtained from New EnglandBiolabs. Taq DNA polymerase is obtained from HT Biotechnology. Pfu DNApolymerase is obtained from Stratagene. Ultrapure dNTP from Pharmacia.Proteases and the protease inhibitor PEFABLOC™ are obtained fromBoehringer Mannheim, except chymotrypsin and trypsin TPCK-treated whichare obtained from Sigma. All other chemical are likewise obtained fromSigma.

Phage Preparation

Escherichia coli TG1 [42] is used for cloning and propagation of phage.TG1 harbouring fd-DOG [43] or derivatives is grown overnight in 2×TYcontaining 15 μg/ml tetracycline. Phagemids are rescued using KM13 orVCSM13 as described [27]. Phage particles are prepared by two PEGprecipitations [44].

Vector Construction

The phage vector fd-DOG [43] is used as parent vector for constructionof the protease cleavable fd-K108. Unique restriction sites (SfiI, KpnI)are introduced into the glycine rich spacer region between D2 and D3using the SCULPTOR™ in vitro mutagenesis system (Amersham) and theoligonucleotide pklinker (Table 4). Further restriction sites (ApaI,SalI) and sequence encoding a protease cleavage site are cloned betweenthe SfiI and KpnI sites using the oligonucleotides polyxafor andpolyxaback to create the vector fd-K108.

The protease cleavable helper phage KM13 is prepared from fd-K108 bytransplanting into the helper phage VCSM13 a BamH1-ClaI fragmentgenerated by PCR and primers fdPCRBack and LIBSEQfor.

A protease cleavable phagemid vector is derived from fd-K108 much asabove except using pCANTAB 3 (Pharmacia). A FLAG-tag is introduced atthe N-terminus of D1 by cloning of a NotI-SfiI fragment generated by PCRand primers Flagprimer and LSPAback. To circumvent deletions due torepeated sequence in the D2-D3 linker, the codon usage of the polylinkerregion is changed in two steps (a) using a Bam-SfiI fragment generatedby PCR and primers RECGLYfor and LIBSEQfor, screening recombinants byPCR and the primers LSPAfor and LSPAback, (b) using a KpnI-ClaI fragmentgenerated by PCR and the primers RECGLYback and LIBSEQback, screeningrecombinants using LSPAfor and LSPAback. The resulting vector is pK1.The entire p3 gene is sequenced using PCR cycle sequencing withfluorescent dideoxy chain terminators (Applied Biosystems) [45]. The“out of frame” vector pK2 is derived from pK1 by site direct mutagenesisusing the oligo delCKpn and the SCULPTOR™ in vitro mutagenesis system(Amersham) kit. The precise sequences of pK1 and pK2 are set forth inKristensen et al., (1998) Folding & Design 3: 321-328.

Cloning of Barnase and Villin

The vectors encoding the single barnase mutants, His102->Ala andLeu14->Ala [29, 46] are used as templates for PCR amplification withprimers Barnasefor and BarnaseH102Aback and Pfu polymerase. The PCRproducts (encoding the single mutant His 102->Ala, and the double mutantHis 102->Ala, Leu14->Ala) are digested using the restriction enzymesSfiI and KpnI, and ligated into vector pK2 to give the phagemids pK2BAand pK2BB respectively and the barnase genes sequenced using PCR cyclesequencing.

The 35 amino acid thermostable fragment of the headpiece of the f-actinbinding protein villin [33] is amplified from chicken bursa cDNA usingPCR primers villinfor and villinback with Pfu polymerase. The PCRproducts are cloned as above to give the phagemid pK2V.

Resistance of Phages to Denaturants, pH and Proteases

For resistance to denaturants, 10 M urea in PBS (25 mM NaH₂PO₄, 125 mMNaCl pH 7.0) or 8 M GndHCl (Guanidine hydrochloride) and 50 mM Tris-HClpH 7.4, 1 mM CaCl₂ (buffer A) is added to 10μ21 phage stocks (10⁸-10¹⁰TU) to give a volume of 1 ml and the conditions specified in Table 1.The phage are incubated for 1-2 hrs, then 100 μl aliquot added to 1 mlTG1 (OD600˜0.5) and serial dilutions plated on TYE plates with 15 μg/mltetracycline. For resistance of phage to extremes of pH (2-12), Trisglycine or Tris HCl buffers (0.1 M glycine or 0.1 M Tris respectively)are added to 10 μl phage stocks, and to neutralise each 100 μl aliquotwe added 50 μl 1 M Tris-HCl pH 7.4 before infection. For resistance totemperature, buffer A is added to 10 μl phage stocks to give a volume of1 ml and incubated at a given temperature (20-60 C.) for 1 hr. 100 μlaliquots are added to TG1 and plated as above. For resistance toproteases, 100 mM NaCl, 50 mM Tris-HCl, 1 mM CaCl₂ pH 7.4 (Factor Xa 100ng/ml or trypsin, chymotrypsin, thrombin, thermolysin and subtilisin all100 μg/ml) or 50 mM Tris-HCl, 1 mM EDTA pH 7.4 (IgA Protease 10 ng/ml)or 50 mM NH₄CO₃ pH 8.0 (Arg-C100 μg/ml, Glu-C 100 μg/ml) or 25 mMNaH₂PO₄, 125 mM NaCl pH 7.0 (AspN 40 ng/ml) is added to 10 μl phagestocks (fd-DOG and fd-K108) to give a volume of 100 μl and a finalconcentration of protease as indicated. Digestions are incubated for 15min at room temperature, samples (100 μl) are then infected into TG1 asabove.

For resistance to proteases in the presence of denaturants samples areprepared as above for urea and temperature denaturation. To 90 glaliquots 10 121 trypsin (1 mg/ml) is added, after 5 mM at roomtemperature 4 A1 PEFABLOC™ protease inhibitor (100 mM) is added and thesamples are infected into TG1 as above.

Western Blot

Phages (pHEN1 rescued using KM13 and pK1 rescued using VCSM13) aresubjected to SDS-PAGE [47] before or after cleavage by trypsin (50ng/ml). After semidry transfer to PVDF membranes the filter is processessentially as described [27]. The primary antibody, monoclonalanti-gill (MoBiTec), is added in a 1:5000 dilution followed byanti-mouse HRP-conjugated antibody (Sigma) in a dilution of 1:50000.Finally the filter is developed using the luminol basedChemiluminescence Western Blotting kit (Boehringer Mannheim).

ELISA

Phage displaying barnase mutants are analysis for binding to the RNaseinhibitor barstar as described [44]. 10 pmol biotinylated barstar ismixed with approximately 10¹⁰ phage displaying barnase mutant A orbarnase mutant B or villin or buffer A. Phage binding barstar iscaptured on Streptavidin coated plates (Boehringer Mannheim) anddeveloped using HRP conjugated anti-M13 antibody (Pharmacia) and2,2′-Azino-Bis(3-Ethylbenzthiazoline-6-sulfonic acid) (Sigma).Absorbance readings are taken at 405 nm.

Temperature Denaturation

At each temperature approximately 10¹⁰ phage displaying the barnasemutants or villin (ampicillin resistant) is mixed with a cleavablecontrol fd-K108 (tetracycline resistant), and a non-cleavable controlphagemid, a chloramphenicol resistant derivative of pHEN1, rescued withKM13 in a total volume of 90 μl of buffer A. After equilibration for20-30 min at the temperature indicated, 10 μl trypsin (5 μg/ml) is addedand the incubation continued for 2 min. Trypsin is neutralised by adding4 μl 100 mM PEFABLOC™ protease inhibitor. Infection and serial dilutionis performed in TG-1 as above and aliquots are plated on TYE platescontaining 100 μg/ml ampicillin+1% glucose, 30 μg/ml chloramphenicol+1%glucose or 15 μg/ml tetracycline.

Selection Experiments

10 μl of serial dilutions of the barnase mutant phage A is mixed with 10μl of the non-diluted barnase mutant phage B in 70 μl buffer A. After 30min incubation at 20° C. or 37° C. 10 μl trypsin (5 μg/ml) is added.Following 2 min. of digestion 4 μl PEFABLOC™ protease inhibitor (100 mM)is added. The phage are infected into TG1 as above. A second round ofselection are performed by scraping bacteria in 3 ml 2×TY, 50 μlinoculated into 50 ml 2×TY/Amp/Glu and the phagemid rescued and phageprepared as above. Clones are analysed by PCR using the primers LSPAforand LSPAback followed by restriction digestion using DdeI.

Selections between pK2V and pK1 phage particles are performed as above,except the selection is performed at 10° C. Clones are analysed by PCRusing the primers LSPAfor and LSPAback.

EXAMPLE 7 Use of Protease-Cleavable Helper Phage for Selection of SignalSequences

Translocation of proteins is directed by signal peptides [48]. These areknown to share common features such as a positively chargedamino-terminal region, a hydrophobic sequence and a carboxy-terminalregion including the signal peptidase cleavage site. Signal peptides areinvolved in “an array of protein-protein and protein-lipid interactions”[49]. The signal sequence may in addition interfere with the proteinfolding pathway. They are also involved in translational regulation,mainly through the downstream box.

Enzymes such as DNA polymerase are very poorly displayed on phageparticles, making their selection almost impossible. In order to improvethe capabilities of phage display to select polymerases, therefore,improved signal sequences are designed and selected for using a phagedisplay technique in which “empty” phage background is eliminated bydigestion of helper phage.

Design of a signal sequence for optimal polymerase display on phage isnot easily achieved. A selection strategy is therefore devised toisolate signal sequences from a library where mutations are introducedat selected sites. Although the signal sequence is not present on phage,its sequence is easily retrieved by sequencing the phagemid locatedwithin the phage particle.

Two libraries are generated from pelB and g3 leader sequences, makinguse of the following oligonucleotide primers for PCR amplification:

1: TACGCCAAGCTTGCATGC; (SEQ ID NO: 18); 2: CTGCACCTGGGCCATGG; (SEQ IDNO: 19); 3: GATTACGCCAAGCTTTG; (SEQ ID NO: 20); 4:GATTACGCCAAGCTTGCATGCANNDDCTNTD (SEQ ID NO: 21);TCAAGGAGACAGTCATAATGARRNNBCTATT GSYAAYRSYASYASYAGBNTTGTTATTACTCSYANYCVNNCYGDCCATGGCCCAGGTGCAGC TG; 5: GATTACGCCAAGCTTTGNNNNCTTTTTTWWG(SEQ ID NO: 22) GAGATTTTCAACRTGARAARATTATTATTCSYAATTSYTTTAGTTSYTSYTTTCTWTGYGGY CCAGCCGGCCATGGCCCAGGTGCA. 6:CTTTATGCTTCCGGCTCG. (SEQ ID NO: 23) 7: CGGCCCCATTCAGATCC. (SEQ ID NO:24)

The restriction sites HindIII and NcoI are noted in italics.

Library I deriving from the pelB leader and library II deriving from theg3 leader are prepared by PCR amplification of 4 (pelB) amplified with 1and 2 and of 5 (g3) amplified with 3 and 2 respectively. Each PCRproduct is digested with HindIII and NcoI and purified with a gelextraction kit (Qiaquick, Qiagen). 0.2 μg of each resulting insert ismixed for ligation with about 2 μg of pHEN1-Stoffel vector (FIG. 6)previously digested with HindIII and NcoI and dephosphorylated withalkaline phosphatase (Boehringer Mannheim). The ligation mixture ispurified by phenol-chloroform extraction and ethanol precipitation priorto electroporation into freshly prepared E. coli TG1.

Randomisation of 32 and 20 bases for the pelB and g3 leadersrespectively is carried out: (i) near and within the Epsilon sequencejust upstream the Shine-Delgamo sequence (ii) downstream theShine-Delgarno sequence near and within the Downstream box [50] (iii)within the leader peptide at the N-terminal region containing thepositively charged amino acid residues, in the hydrophobic region [51]and in the C-terminal region close to the highly conserved peptidasecleavage site [52].

Phage is produced as described previously [25] except that the helperphage KM13 (Example 3) is used instead of VCSM13 and that 0.1 mM IPTG isadded when specified to the overnight culture at 30° C. Selections forresistance to trypsin and for binding [44] is done as described earlier,except that 11 μg of anti-Taq antibody (Taqstart, Clontech) is coatedovernight on immunotubes (Nunc). PCR screening is done with primers 6and 7 using single E. coli TG1 colonies containing the phagemid astemplate following a previously described protocol; after gelelectrophoresis on a 2% agarose gel. The size of the amplified fragmentis used as a criterion to establish whether deletions within thepolymerase gene have occurred.

The calculated diversity of the libraries computed from the degeneracyof the synthesised oligonucleotides is about 3.7×10¹³=4⁹×3⁷×2¹⁶ and1.7×10⁷=44×216 for the pelB and g3 leaders respectively. Aftertransformation of E. coli with the phagemid libraries, the library sizemeasured as the number of ampicillin-resistant colonies is found to be1.3×10⁷ and 9.6×10⁶ for the pelB and g3 leaders respectively.

The selection for display of the polymerase is done by cleavingspecifically the helper phage p3 copies with the protease trypsin so asto render non-infective all phage particles that are not expressing anyp3-polymerase fusion protein.

Both libraries are mixed and the selection rounds are carried out in twoconditions, with or without 0.1 mM IPTG in the culture medium. WithIPTG, deletions of the polymerase gene or of part of it are noticedafter round III (4 out of 28 clones) as shown by a PCR screening (seeabove); after round IV, these clones represent most of the population(28 out of 30). Without IPTG, these clones represent a significant partof the selected ones after round VI (3 out of 12). The selection istherefore changed from round five on by introducing in addition toselection for protease resistance, a selection for binding to ananti-Taq antibody. After the selection rounds VII and VIII, (3 out of13) and (0 out of 19) clones respectively correspond to deletedp3-polymerase fusion proteins.

For characterisation of the leaders, the HindIII-NcoI fragments aresubcloned after PCR amplification of individual E. coli colonies. Theresulting phagemids are noted pHEN1-1x/Stoffel subcloning with x=7, 9,10 and 12. The HindIII-NcoI and the NcoI-NotI fragment corresponding tothe Stoffel fragment is sequenced on both strands using a 373A DNAsequencer (Applied Biosystems).

For ELISA, an anti-Taq antibody (Taqstart, Clontech) is used for coatingthe ELISA plate and an anti-M13-horseradish peroxidase fusion protein(Pharmacia Biotech) is used for detection in a standard protocol [44].

Expression of polymerase in the supernatant is made by infection of E.coli HB2151 with selected phagemids [25, 27] except that the IPTGconcentration is 0.1 mM instead of 1 mM. About 10 μl of supernatant isloaded on a polyacrylamide gel for electrophoresis (Novex); the gel isblotted onto nitrocellulose (Protran, Schleicher and Schuell) and ananti-Taq polymerase antibody (Taqstart, Clontech), and a goat anti-mouseIgG-horseradish peroxidase (Sigma) prior to detection on autoradiographyfilms by chemiluminescence (ECL™ chemiluminescence reagents, Amersham).

Four individual clones 7, 9, 10 and 12 from round VII, that werescreened for protease resistance among 12 clones, are furthercharacterised. To ensure that only mutations within the signal sequenceare considered, and not mutations somewhere else within the phagemidthat may have occurred during amplification at the various rounds, thesignal sequences are subcloned into the original vector. The resultsshown in Table 5 indicate that optimal polymerase display for theselected clone 10 is about 50 fold higher than for the originalsequence. This result is confirmed independently within experimentalerrors by an ELISA using anti-Taq antibody and anti-M13-HRP: 10⁹ phageparticles of the pelB leader phagemid pHEN1-Stoffel (signal to noiseratio: 1.46) give an identical signal as 10⁷ phage particles of clone 10(signal to noise ratio: 1.47).

The expression of Stoffel fragment in E. coli HB2151 is also studied forthe various leaders (see Table 6). The concentration of Stoffel fragmentin the culture supernatant is estimated by comparing the spotintensities for known amounts of polymerase and found to be about 0.1mg/l for the pelB leader. A 3-fold increase in expression is observedfor the leaders 17 and 110, whereas an about 3-fold decrease is notedfor leader 19.

Table 5. Number of Phage-Polymerases per Phage Particle with LeaderpelB, as a Function of Temperature and IPTG Concentrations.

The phage titer is measured as the number of infective phage particlesand the phage-polymerase titer as the number of infective phageparticles after treatment with trypsin. The number of phage-polymerasesper phage particle is the ratio of the titers. As the phage particleswere rescued with a helper phage, phage displays either a p3-polymerasefusion protein and a few p3 copies containing a trypsin-cleavage site oronly these p3 copies.

TABLE 6 Phage characterisation for leader pelB or selected leaders fromround VII for optimal polymerase display Temperature 25° C. 30° C. 37°C.   0 mM IPTG 1.7 × 10⁻³ 9.1 × 10⁻⁴ 1.2 × 10⁻³ 0.1 mM IPTG 9.1 × 10⁻³ *8.3 × 10⁻³ 2.4 × 10⁻³ *   1 mM IPTG 1.5 × 10⁻² * 5.5 × 10⁻³ * 1.2 ×10⁻³ * (same legend as for Table 5; culture in 2xTY at T = 30° C.without IPTG). * in these conditions, the phage titer drops below1010/m1 of culture medium.

TABLE 7 Randomised and selected sequences. Number of phage-polymerasesLeader Titer × 10¹¹ per phage particle pelB 1.2 9.1 × 10−⁴ 17 2.0 2.5 ×10−² 19 0.5 8.3 × 10−³ 110 0.7 4.3 × 10−² 112 1.6 1.4 × 10−²

The randomised DNA sequence is given from 5′ to 3′; above and below it,the bases that differ from the given sequence in the signal sequencespelB, 17, 19, 110 and 112 are indicated. The Shine-Delgamo sequence, thestart codon and the last codon of the signal sequence, GCC, have beenunderlined. The HindIII and the NcoI restriction sites are in italics.The corresponding amino acid sequences are given below. Library I isinitially designed from the pelB leader and library II from the g3leader.

III-A. From library I pe1B                 AATT     A   T        AATAC(SEQ ID NO: 25) 5′ AAGCTTGCATGCANNDDCTNT DTCAAGGAGACAGTCATAATGARRNNB CT(SEQ ID NO: 26) 17                 GCAT     C   G    AGACG (SEQ ID NO:27) 110                 CGGG     G   T    GAGGG (SEQ ID NO: 28) 112                CCAG     C   T    GGCGG (SEQ ID NO: 29) pe1B    CCT CGGCGCCGCT GA    GCGGC CAG    C    G (SEQ ID NO: 30)  ATTGSYAAYRSYASYASYAGBNTTGTTATTACTC  SYANY CVNNCYGDCCAT GG CC 3′ (SEQID NO: 31) 17     GC   TGGT    CT   GT       GA  CC   CC   GGT C    T(SEQ ID NO: 32) 110    GC   TGCT    GT   GC       GG  CC   AT   GCG C    G (SEQ ID NO: 33)112     GT   TAGC    GC   GT       GG  CT   GC   CCC C    A (SEQ ID NO:34) pe1B       M K Y L L P T A A A G L L L L A A Q P A M A (SEQ ID NO:35) 17       K T   A M V L V G        P P G P S (SEQ ID NO: 36) 110      R G   A M L V A G        P I    A P A (SEQ ID NO: 37) 112       RR   V     I      A A V G      L A P P T (SEQ ID NO: 38) III-B. Fromlibrary II g3 leader            GAGC     TT G A A (SEQ ID NO: 39)5′ AAGCTTTGNNNNCTTTTTTWWGGAGATTTTCAACRTGARAARATTATTAT (SEQ ID NO: 40) 19           GGGC     TA  A G G (SEQ ID NO: 41) GC    CC   GT CC    A   CC (SEQ ID NO: 42) TCSYAATTSYTTTAGTTSYTSYTTTCTWTGYGGYCCAGCCGGCCATG G CC3′ (SEQ ID NO: 43) 19 CT   CC  GT GC     A   T T (SEQ ID NO: 44) g3leader          M K K L L F A I P L V V P F Y A A Q P A M A (SEQ ID NO:45) 19          R R         L P           V A     Y V V (SEQ ID NO: 46)

EXAMPLE 8 Selection of a Catalytic Activity Using Protease-CleavableHelper Phage

A strategy for the selection of catalysts by phage display is based onselection of the reaction product of a catalytic reaction, and the useof proximity effects to select the catalyst. In this strategy, a taggedsubstrate is crosslinked to the phage in the proximity of the displayedenzyme; the phage is thereby attached to a solid-phase and released byan intramolecular cleavage reaction catalysed by the displayed enzyme[53].

A similar approach has been applied to the selection of active DNApolymerase variants. The approach involves two chemically independentreactions, the catalytic reaction leading to a product (in this casedistinguished by incorporation of a biotin tag) and a chemicalcrosslinking reaction by which the substrate (and product) are linked tothe phage. Selection of the phage by streptavidin beads thereforeselects for phages which are chemically attached to tagged product;these reactions are more likely to be coupled on the same phage asreactions in cis are favoured over reactions in trans by proximity.

Maleimides are used in a chemical cross-linking reaction. These areknown to react with thiols and in alkaline solutions with amino groups,and are therefore capable of reacting with a wide range of sites on thephage and on the displayed enzyme. A covalent product between the majorcoat protein (p8) and N-biotinoyl-N′-(6-maleimidohexanoyl) hydrazide, isdetected by SELDI mass spectrometry. Two amino groups (the N-terminalAla-1 and the residue Lys-8) are thought to be involved.

The strategy is tested using DNA polymerases in view of their centralrole in molecular evolution. A maleimidyl group is introduced at the 5′end of a DNA primer; the product is tagged by addition of biotinylateddUTP to the 3′ end of the primer by the catalytic action of thepolymerase. The Klenow and Stoffel fragments of DNA polymerase IEscherichia coli and Thermus aquaticus, respectively, are cloned fordisplay by fusion to the pIII coat protein of filamentous bacteriophageby conventional techniques. Both fragments lack the 5′ to 3′ exonucleasedomains; the Stoffel fragment also lacks a 3′ to 5′ exonucleaseactivity.

The fusion protein is cloned on a phagemid (pHEN1) [25], and is rescuedby a helper phage. The polymerase fragments are shown to be displayed onthe phage (after rescue with helper phage) by binding of the phage towells coated with anti-polymerase antibodies as detected by ELISA (notshown). The phage are also analysed by Western blot using anti-p3 oranti-polymerase antibodies. This confirms the presence of the fusionprotein, but also indicates contamination by free polymerase. Presumablythis arises by secretion from the bacterial host through incompletesuppression of the amber stop codon or by cleavage from the phagesurface. This is removed by a further step of ultracentrifugation or bysize exclusion chromatography. The purified phages are assayed for DNApolymerase activity in a primer/template extension assay withradioactively labelled α³²P-dCTP and found to be active.

However as is indicated by the Western blots, the polymerase-p3 fusionprotein is poorly incorporated into the phage compared to the p3protein. This appears to be due to incorporation of p3 from the helperphage, as shown by the alternative use of a helper phage (KM13) in whichthe p3 protein of the helper phage (but not that of fusion protein) canbe cleaved with trypsin so as to render it incapable of mediatinginfection (Examples 3 and 4). Thus after proteolysis only those phagesthat had incorporated the fusion protein are infective; from the loss intitre after proteolysis we estimate that only one phage particle in athousand had incorporated the fusion protein. The selection process,relying on tagging by polymerase in cis, would be compromised by such agreat excess of phages lacking the polymerase but available for taggingin trans. Selected phages are therefore treated with trypsin to destroythe infectivity of those lacking the displayed polymerase.

The phage displaying the Stoffel fragment are incubated with primer 13[TTT CGC AAG ATG TGG CGT] (SEQ ID NO: 47) comprising a 5′ maleimidylgroup and a 3′ biotinylated nucleotide. After incubation the phage arecaptured on streptavidin-coated beads, with a yield of about 1-5% ofinfectious phage. This shows that primer can be chemically cross-linkedto the phage, presumably via p8 protein as shown for theN-biotinoyl-N′-(6-maleimidohexanoyl) hydrazide. The phage are thenincubated with primer 1b [GCGAAGATGTGG] (SEQ ID NO: 48) comprising a 5′maleimidyl group in the presence of biotin-dUTP 2 and template 3 [AAATAC AAC AAT AAA ACG CCA CAT CTT GCG] (SEQ ID NO: 49). Capture of thephage is dependent on presence of 1b, 2 and 3 (Table 8), but also on theinclusion of trypsin, which cleaves the helper phage to reducenon-specific phage isolation.

TABLE 8 Selection of catalytically active phage-Stoffel particles.φi^([a]) φf Yield in tu In tue In % Conditions^([c]) 8.4 × 10⁵ 2.0 × 10⁴2.4 3.6 × 10⁵ 1.0 × 10² 0.028 primer 1b 4.4 × 10⁵ 3.0 × 10² 0.068biotinylated dUTP 2 4.8 × 10⁵ 3.0 × 10² 0.062 template 3 4.4 × 10⁹ 4.0 ×10⁶ 0.091 trypsin 1.5 × 109 5.5 × 105 0.037 trypsin, primer 1b φi and φfdenote the number of transformation units (tu) prior ^([a])and after^([b])the selection. Yield = φf/φi. ^([c])+ primer 1b, + biotinylateddUTP 2, + template 3 and + trypsin.

EXAMPLE 9 Selection for Disulphide-Containing Polypeptides

For the cloning of (poly)-peptide encoding DNA fragments and theirdisplay for selection between barnase and p3, the phage fd-3 isconstructed (FIG. 5). Phage fd-3 comprises the H102A mutant of barnaseN-terminally fused to the p3 gene of phage fd-TET. Between the codon forthe last residue of barnase and the first residue of p3 is thenucleotide sequence CTG CAG GCG GTG CGG CCG CA (SEQ ID NO: 50). Thissequence contains a PstI DNA restriction site (in italics) for insertionof DNA fragments flanked by PstI restriction sites. The sequence furtherintroduces a frame shift between barnase and p3, which preventsexpression of the correct p3 reading frame in fd-3. Phage particles ofphage fd-3 therefore do not display the infection protein p3 and arenon-infectious.

Phage fd-3 is therefore well suited as a cloning vector as vectorswithout PstI DNA inserts after ligation are not propagated duringselection. Statistically 1 out of 3 random DNA inserts in the PstIrestriction site will (except for the presence of stop-codons within theinsert) create an open reading frame spanning barnase, the insert itselfand p3 and result in infectious phage particles containing p3 in thephage coat. In these recombinant clones barnase is followed by theinsert, which is then followed by the amino acid residues AGGAAA (SEQ IDNO: 80) before the start of the p3 protein. This AGGAAA (SEQ ID NO: 80)peptide should provide enough flexibility in the fusion protein toenable the infectivity function of p3 and the access of the protease tothe N-terminal appendices of p3.

Genomic DNA from the E. coli strain TG1 is amplified in 30 cycles of apolymerase chain reaction (PCR) with an annealing temperature of 48° C.using the oligonucleotide SN6MIX (5′-GAG CCT GCA GAG CTC AGG NNN NNN-3′)(SEQ ID NO: 51), which comprises 6 degenerate positions at theextendible 3′ end to ensure random priming. In a second step of 30 PCRcycles with an annealing temperature of 52° C. primary PCR products areextended by re-amplification with the oligonucleotide XTND (5′-CGT GCGAGC CTG CAG AGC TCA GG-3′) (SEQ ID NO: 52). Products with a length ofaround 150 bp from this reaction are purified from an agarose gel andreamplified in 30 PCR cycles using an annealing temperature of 52° C.and the oligonucleotide XTND. These reamplified 150 bp fragments arepartially digested with SacI (site indicated in bold in theoligonucleotides) and ligated for dimerisation. Ligated products arereamplified in a further 10 PCR cycles with an annealing temperature of44° C. followed by a 30 PCR cycles with an annealing temperature of 55°C. using the oligonucleotide XTND. The annealing temperatures are chosento discriminate against priming of the oligonucleotide in the middle ofthe dimerised fragments. The reaction product is size purified twice onan agarose gel to remove monomers and oligomers (non-dimers).

The final dimer fraction is amplified by PCR using an annealingtemperature of 55° C. and the oligonucleotide XTND on a large scale,digested with PstI (site indicated in italics in oligonucleotides) andligated into the also PstI digested and phosphatased vector fd-3. Afterelectroporation into E. coli bacteria a repertoire of 3.6×10⁷recombinants is obtained. Sequence analysis of randomly picked clonesreveal the presence of mainly dimeric (11 out of 14) and some monomeric(2 out of 14) DNA inserts.

Reinfection of E. coli bacteria with phage produced from the initialpopulation of transformed cells yields, according to sequence analysisof twenty randomly picked clones, a library of infectious phagescontaining almost exclusively barnase-(in-frame,no-stop-dimer-insert)-p3 fusions. Non-infectious phages arising fromvector without insert and from vector with out-of-frame or stop-codoncontaining inserts are not propagated in the infection step. The vectorfd-3 is therefore suitable to create a repertoire of polypeptidesrandomly generated through dimerisation of DNA fragments from the E.coli genome.

This repertoire of polypeptides displayed as an inserted fusion betweenbarnase and p3 on phage fd is subjected to proteolytic digestion withtrypsin and thermolysin alone or a mixture of both (1 ng/μl each) inTBS-Ca buffer (25 mM Tris, 137 mM NaCl, 1 mM CaCl₂, pH 7.4). Afterproteolysis phage is captured with biotinylated barstar bound to aStreptavidin coated microtitre well plate and eluted at pH 2.0. Phage isneutralised to pH 7 and propagated through reinfection of the E. colicells and selected for a second round as before. All steps are performedin the absence of any reducing agent like dithiothreitol (DTT) orβ-mercaptoethanol, which would reduce and thereby cleave any potentialdisulphide bonds formed between cysteine sidechains within selectedpolypeptides.

Randomly picked phages, which are eluted after the first and secondround of proteolytic selection, are analysed for binding to barstarafter incubation with a mixture of trypsin and thermolysin under theconditions of the selection (Table 9). Their sequence is determined(Table 9). 17 out of 18 analysed clones treated with a mixture oftrypsin and thermolysin during selection are found to bind biotinylatedbarstar after incubation with trypsin and thermolysin. 5 out of 8analysed clones treated with trypsin during selection bound biotinylatedbarstar after incubation with trypsin and thermolysin, whilst 9 of 14analysed clones treated with thermolysin are found to bind. No randomlypicked clones not treated with a protease during selection bindbiotinylated barstar after incubation with trypsin and thermolysin.Binding to biotinylated barstar shows that the polypeptide insertionbetween barnase and p3 on the phage retains its overall covalentintegrity and therefore keeps the N-terminal tag (barnase) and theinfection protein p3 (and thereby the phage particle as a whole)covalently linked.

However, the possibility of proteolytic digestion of the peptidebackbone can not be excluded, as the inserts may also be kept covalentlylinked through bonds between sidechain groups like the SH₂ groups ofcysteines. Sequence analysis of the selected clones reveal that 19 of 25(76%) resistant clones contain two or more cysteine residues.

To analyse the role of possible disulphide bonds in the polypeptideinserts, 13 of the selected phages are analysed for binding tobiotinylated barstar (and thereby for a covalent linkage of barnase andp3 through the insert) after treatment with trypsin and thermolysinfollowed by a wash with 20 mM DTT before detection of bound phage. 2phage clones, which bind barstar after protease treatment without DTTwash and contain no cysteines, are unaffected by the DTT treatment.Another 2 phage clones, which bind barstar after protease treatmentwithout DTT and contain two or more cysteines, are also observed to bindbarstar after protease and DTT treatment. This suggests that theirinserts are protected from proteolysis of their peptide backbone despitethe presence of principle substrate sites for the proteases.

These inserts are therefore protected from proteolytic attack due to aconformational restraint of their peptide backbone. However, 9 phageclones which bind barstar after protease treatment without DTT wash andcontain two or more cysteines are observed not to bind barstar afterprotease and DTT treatment. This suggests that their inserts areproteolytically cleaved in their peptide backbone, but are held togetherby disulphide bonds between cysteine sidechains in the absence of thereducing agent DTT. These inserts are therefore not protected fromproteolytic attack due to a conformational restraint of their peptidebackbone.

Thus, the method of the present invention may be configured to selectfor cysteine-containing polypeptides, even where the polypeptides wouldby susceptible to protease attack since the polypeptides are capable ofbeing held together in the selection step by disulphide bonds.

Table 9. Barstar binding of phages displaying barnase-p3 fusion insertsselected after proteolytic treatment under non-reducing conditions andamino acid sequences of their PstI inserts in vector fd-3.

Barstar binding (−DTT) after proteolysis of phage with trypsin andthermolysin (2 ng/μl each) without a 20 mM DTT wash is determined bymeasurement of a signal that is at least 60% of the signal for barstarbinding of phage without protease treatment. Barstar binding (+DTT)after a 20 mM wash is determined by measurement of a signal that is atleast 60% of the signal for barstar binding (−DTT) without a 20 mM DTTwash. Phage are randomly picked after one (1×) or two (2×) rounds ofproteolysis with trypsin (Tr) or thermolysin (Th). Phages are treatedwith proteases, captured with biotinylated barstar microtitre wellplates, and washed with 20 mM DTT where applicable. Bound phage aredetected with a horse radish peroxidase conjugated anti-M13 phageantibody (Pharmacia Biotech).

Proteo- lytic Phage selec- Barstarbindg Amino acid sequence clone tion−DTT +DTT of inserts TA-1.2 1xTr yes no LQSSGDCVIS DTCIAGMAEA AACEEKFSSQNVGLTITVTP CLSSA (SEQ ID NO: 53) TA-2.25 2xTr yes no LQSSGCGSSGSSINCLPCGA TSRGTSPLAS GLPSSATIHC LSSA (SEQ ID NO: 54) TA-2.26 2xTr yesno LQSSGDSAGC KNMTGGRLYA HTLEAIIPGF AVSAPACEPA (SEQ ID NO: 55) TA-2.272xTr yes yes LQSSGCVRLK RTSVNHQPDA WPEPHLKAAC EPA (SEQ ID NO: 56)TA-2.30 2xTr yes no LQSSGCGSSG SSINCLPCGA TSRGTSPLAS GLPSSATVQC LSSA(SEQ ID NO: 57) TB-1.10 1xTh yes yes LQSSGKIVQA GANIQDGCIM HGYCDTDTIVGENGHIGLSS A (SEQ ID NO: 58) TB-1.11 1xTh yes yes no insert, Barnase &p3 in frame TB-2.33 2xTh yes no LQSSGVCVIS DTCIAGTAEA AACEEKFSSQNVGHTITETP CLSSA (SEQ ID NO: 59) TB-2.34 2xTh yes no LQSSGCGSSGSSINCLPCGA TSRGTSPLAS GLPSSATIQC LSSA (SEQ ID NO: 60) TB-2.35 2xTh yesno LQSSGQDSQR EHASHTAEDD CEDQTRIHQH IREVDFVDTP QEVDDCRAAL SSA (SEQ IDNO: 61) TB-2.37 2xTh yes no LQSSGCVRLK RTSVNHQPDA WPEPHLKAAC EPA (SEQ IDNO: 62) TB-2.38 2xTh yes yes LQSSGVRPA (SEQ ID NO: 63) TB-2.39 2xTh yesno LQSSGCGSS GSSINCLPCGA TSRGTSPLAS GLPSSATIQ CLSSA (SEQ ID NO: 64)

EXAMPLE 10 Use of Reducing Agents to Eliminate Disulphide-RelatedBackground

The repertoire of polypeptides described in Example 9 is digested asbefore with both trypsin and thermolysin (Example 9) except for anadditional washing step here with 50 mM DTT after binding of theproteolytically treated phage to the microtitre well plates coated withStreptavidin-biotinylated barstar. This washing step is designed to washoff phage displaying an N-terminal barnase tag, which is no longerlinked to p3 through an intact polypeptide backbone but only throughdisulphide bonds between cysteine sidechains in the polypeptide insertsbetween the barnase tag and p3.

12 randomly picked phages eluted after the second round of proteolysisare analysed for stability against a mixture of trypsin and thermolysinunder the conditions of the selection and their sequence is determined(Table 10). 10 clones treated with a mixture of trypsin and thermolysinduring selection bind biotinylated barstar after incubation with trypsinand thermolysin followed by washing with 50 mM DTT before detection ofcaptured phage. Only one of these clones contain two or more cysteines.

Proteolytic treatment of the phage library followed by a wash with DTTtherefore allows the selection of peptide inserts which are protectedfrom proteolysis and which are not held together through disulphidebonds.

Table 10. Barstar binding of phages displaying barnase-p3 fusion insertsselected after proteolytic treatment followed by treatment with 50 mMDTT and amino acid sequences of their PstI inserts in fd-3. Barstarbinding (+DTT) after proteolysis of phage with trypsin and thermolysin(2 ng/μl each) followed by a 50 mM DTT is determined by measurement of asignal for Barstar binding (+DTT), which is at least 60% of the signalfor barstar binding of phage without protease treatment. Phage arerandomly picked after two (2×) rounds of proteolytic with trypsin (Tr)and/or thermolysin (Th). Phages are treated with proteases, capturedwith biotinylated barstar in microtitre well plates, and washed with 50mM DTT where applicable. Bound phage is detected with a horse radishperoxidase conjugated anti-M13 phage antibody.

Proteo- lytic Phage selec- Barstarbindg Amino acid sequence Clone tion+DTT of inserts B2-13 2xTr/Th yes LQSSGTEVDR GNQQHDTNDR DFTHTPLSS A (SEQID NO: 65) B2-14 2xTr/Th yes LQSSGSVAQG SSASVDVTAT NAVLSADSL SLGGGEPA(SEQ ID NO: 66) B2-22 2xTr/Th yes LQSSGGAVAV TPGPVLSSA (SEQ ID NO: 67)B2-23 2xTr/Th yes LQSSGHCRGK PVLCTHTA (SEQ ID NO: 68) B2-15 2xTr/Th yesLQSSGVRPA (SEQ ID NO: 69) B2-17 2xTr/Th yes no insert, Barnase & p3 inframe B2-20,21 2xTr/Th yes no insert, Barnase & p3 in frame B2-16,242xTr/Th yes LQSSGEPAPA HEAKPTEAPV AKAEAKPETP AHLSSA (SEQ ID NO: 70)B2-18 2xTr/Th no LQSSGCVRLK RTSVNHQPDA WPEPHLKAAC EPA (SEQ ID NO: 71)B2-19 2xTr/Th no LQSSGVVDWA KMREIADSIG AYLFVDMAHV AALSSA (SEQ ID NO: 72)

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Cleavage of phages with protease sites. Phages were prepared byrescue with KM13 (pHEN1, A+B), or with VCSM13 (pK1, C+D). Uncleaved(A+C) or cleaved with trypsin (B+D). 5 μl 2.5 μl and 1 μl phages wereloaded as indicated. Molecular weight markers are in kD.

FIG. 2. The phagemid vectors pK1 and pK2. These vectors contain aprotease cleavable sequence between D2 and D3 of the phage p3 protein.In pK1 (SEQ ID NO: 73 and SEQ ID NO: 74, respectively), D2+D3 are inframe; in pK2 (SEQ ID NO: 75 and SEQ ID NO: 76, respectively), D3 is outof frame.

FIG. 3. Binding of phage-barnase to barstar. Phage displaying differentfusion protein are incubated with biotinoylated barstar captured onstreptavidin-coated plate and detected by ELISA. a) barnase mutant A, b)barnase mutant B, c) villin, d) no phage.

FIG. 4. Temperature denaturation of phage fusion proteins. Phagemidswere rescued with KM13, infectivity (TU/rnl) shown after incubation andcleavage with trypsin at given temperatures. Fusion with villinsubdomain (triangles), barnase mutant A (diamonds), barnase mutant B(squares), pHEN1-chloramphenicol resistant (circles).

FIG. 5. The fd vector fd-3. The gene for the H102A mutant of Barnase isintroduced by subcloning into fd-DOG [43] after PCR amplification withsuitable oligonucleotides using the restriction sites ApaLI (at theBarnase 5′ end) and NotI to create fd-3. The nucleotide and amino acidsequence of the junction between Barnase and p3 sequences is shown inexpanded view (SEQ ID NO: 77 and SEQ ID NO: 78, respectively).

FIG. 6. Map of phagemid vector used for display of Stoffel fragment onthe surface of phage. The asterisk shows sequences that are randomisedat least partially in libraries I and II.

REFERENCES

-   1. Rubingh, D. N. (1997). Protein engineering from a bioindustrial    point of view. Current Opinion in Biotechnology. 8, 417-422.-   2. Fersht, A. R. (1993). Protein folding and stability: the pathway    of folding of barnase. FEBS Letters. 325, 5-16.-   3. Zhao, H., et al. (1998). Molecular evolution by staggered    extension process (StEP) in vitro recombination. Nature    Biotechnology. 16, 258-261.-   4. Patten, P. A., R. J. Howard, and W. P. C. Stemmer. (1997).    Applications of DNA shuffling to pharmaceuticals and vaccines.    Current Opinion in Biotechnology. 8, 724-733.-   5. Sauer, R. T. (1996). Protein folding from a combinatorial    perspective. Folding & Design. 1, R27-R30.-   6. Munson, M., et al. (1996). What makes a protein a protein?    Hydrophobic core designs that specify stability and structural    properties. Protein Science. 5, 1584-1593.-   7. Dahiyat, B. I., C. A. Sarisky, and S. L. Mayo. (1997). De Novo    Protein Design: Towards Fully Automated Sequence Selection. Journal    of Molecular Biology. 273, 789-796.-   8. Riddle, D. S., et al. (1997). Functional rapidly folding proteins    from simplified amino acid sequences. Nature Structural Biology.    4(10), 805-809.-   9. Hoogenboom, H. R. and G. Winter. (1992). By-passing Immunisation.    Human Antibodies from Synthetic Repertoires of Germline VH Gene    Segments Rearranged in Vitro. Journal of Molecular Biology. 227,    381-388.-   10. Winter, G., et al. (1994). Making Antibodies by Phage Display    Technology. Annual Review of Immunology. 12, 433-455.-   11. Braisted, A. C. and J. A. Wells. (1996). Minimizing a binding    domain from protein A. Proc. Natl. Acad. Sci. USA. 93, 5688-5692.-   12. O'Neil, K. T., et al. (1995). Thermodynamic Genetics of the    Folding of the B1 Immunoglobulin-Binding Domain From Streptococcal    Protein G. Proteins: Structure, Function, and Genetics. 21, 11-21.-   13. Gu, H., et al. (1995). A phage display system for studying the    sequence determinants of protein folding. Protein Science. 4,    1108-1117.-   14. Hubbard, S. J., F. Eisenmenger, and J. M. Thornton. (1994).    Modeling studies of the change in conformation required for cleavage    of limited proteolytic sites. Protein Science. 3, 757-768.-   15. Fontana, A., et al. (1997). Probing the partly folded states of    proteins by limited proteolysis. Folding & Design. 2, R17-R26.-   16. Kamtekar, S., et al. (1993). Protein Design by Binary Patterning    of Polar and Nonpolar Amino Acids. Science. 262, 1680-1685.-   17. Davidson, A. R. and R. T. Sauer. (1994). Folded proteins occur    frequently in libraries of random amino acid sequences. Proc. Natl.    Acad. Sci. USA. 91, 2146-2150.-   18. Davidson, A. R., K. J. Lumb, and R. T. Sauer. (1995).    Cooperatively folded proteins in random sequence libraries. Nature    Structural Biology. 2(10), 856-864.-   19. Matthews, D. J. and J. A. Wells. (1993). Substrate Phage:    Selection of Protease Substrates by Monovalent Phage Display.    Science. 260, 1113-1117.-   20. Riechmann, L. and P. Holliger. (1997). The C-Terminal Domain of    To1A Is the Coreceptor for Filamentous Phage Infection of E. coli.    Cell. 90, 351-360.-   21. Smith, G. P. (1985). Filamentous Fusion Phage: Novel Expression    Vectors That Display Cloned Antigens on the Virion Surface. Science.    228, 1315-1317.-   22. Krebber, C., et al. (1997). Selectively-infective Phage (SIP): A    Mechanistic Dissection of a Novel in vivo Selection for    Protein-ligand Interactions. Journal of Molecular Biology. 268,    607-618.-   23. Stengele, I., et al. (1990). Dissection of Functional Domains in    Phage fd Adsorption Protein. Discrimination between Attachment and    Penetration. Journal of Molecular Biology. 212, 143-149.-   24. Gray, C. W., R. S. Brown, and D. A. Marvin. (1981). Adsorption    complex of Filamentous fd virus. Journal of Molecular Biology. 146,    621-627.-   25. Hoogenboom, H. R., et al. (1991). Multi-subunit proteins on the    surface of filamentous phage: methodologies for displaying antibody    (Fab) heavy and light chains. Nucleic Acids Research. 19, 4133-4137.-   26. Bass, S., R. Greene, and J. A. Wells. (1990). Hormone Phage: An    Enrichment Method for Variant Proteins With Altered Binding    Properties. Proteins. 8, 309-314.-   27. Nissim, A., et al. (1994). Antibody fragments from a “single    pot” phage display library as immunochemical reagents. The EMBO    Journal. 13, 692-698.-   28. Marzari, R., et al. (1997). Extending filamentous phage host    range by the grafting of a heterologous receptor binding domain.    Gene. 185, 27-33.-   29. Mossakowska, D. E., K. Nyberg, and A. R. Fersht. (1989). Kinetic    Characterisation of the Recombinant Ribonuclease from Bacillus    amyloliquefaciens (Barnase) and Investigation of Key Residues in    Catalysis by Site-Directed Mutagenesis. Biochemistry. 28, 3843-3850.-   30. Meiering, E. M., L. Serrano, and A. R. Fersht. (1992). Effect of    Active Site Residues in Barnase on Activity and Stability. Journal    of Molecular Biology. 225, 585589.-   31. Serrano, L., et al. (1992). The Folding of an Enzyme. II    Substructure of Barnase and the Contribution of Different    Interactions to Protein Stability. Journal of Molecular Biology.    224, 783-804.-   32. McKnight, C. J., P. T. Matsudaira, and P. S. Kim. (1997). NMR    structure of the 35-residue villin headpiece subdomain. Nature    Structural Biology. 4(3), 180-184.-   33. McKnight, C. J., et al. (1996). A Thermostable 35-Residue    Subdomain within Villin Headpiece. Journal of Molecular Biology.    260, 126-134.-   34. Xu, D. and R. Nussinov. (1997). Favorable domain size in    proteins. Folding & Design. 3, 11-17.-   35. Kippen, A. D. and A. R. Fersht. (1995). Analysis of the    Mechanism of Assembly of Cleaved Barnase from Two Peptide Fragments    and Its Relevance to the Folding Pathway of Uncleaved Barnase.    Biochemistry. 34, 1464-1468.-   36. Gay, G. d. P. and A. R. Fersht. (1994). Generation of a Family    of Protein Fragments for Structure-Folding Studies. 1. Folding    Complementation of Two Fragments of Chymotrypsin Inhibitor-2 Formed    by Cleavage at Its Unique Methionine Residue. Biochemistry. 33,    7957-7963.-   37. Wu, L. C., R. Grandori, and J. Carey. (1994). Autonomous    subdomains in protein folding. Protein Science. 3, 369-371.-   38. Kwon, W. S., N. A. D. Silva, and J. T. Kellis. (1996).    Relationships between thermal stability, degradation rate and    expression yield of barnase variants in the periplasm of Escherichia    coli. Protein Engineering. 9(12), 1197-1202.-   39. Axe, D. D., N. W. Foster, and A. R. Fersht. (1996). Active    barnase variants with completely random hydrophobic cores. Proc.    Natl. Acad. Sci. USA. 93, 5590-5594.-   40. Waldburger, C. D., J. F. Schildbach, and R. T. Sauer. (1995).    Are buried salt bridges important for protein stability and    conformational specificity? Nature Structural Biology. 2(2),    122-128.-   41. Roy, S., et al. (1997). A Protein Designed by Binary Patterning    of Polar and Nonpolar Amino Acids Displays Native-like Properties.    Journal of the American Chemical Society. 119, 5302-5306.-   42. Gibson, T. J., Studies on the Epstein-Barr Virus Genome. 1984,    Univ. of Cambridge, Cambridge, UK:-   43. Clackson, T., et al. (1991). Making antibody fragments using    phage display libraries. Nature. 352, 624-628.-   44. McCafferty, J., et al. (1990). Phage antibodies: filamentous    phage displaying antibody variable domains. Nature. 348, 552-554.-   45. Fisch, I., et al. (1996). A strategy of exon shuffling for    making large peptide repertoires displayed on filamentous    bacteriophage. Proc. Natl. Acad. Sci. USA. 93, 7761-7766.-   46. Matouschek, A., et al. (1989). Mapping the transition state and    pathway of protein folding by protein engineering. Nature. 340,    122-126.-   47. Laemmli, U. K. (1970). Cleavage of structural proteins during    the assembly of the head of bacteriophage T4. Nature. 227, 680-685.-   48. Schatz, G. and Dobberstein, B. (1996) Common principles of    protein translocation across membranes. Science. 271, 1519-1526.-   49. Von Heijne, G. (1998) Life and death of a signal peptide.    Nature. 396, 111-113.-   50. Sprengart, M. L., Fuchs, E. and Porter, A. G. (1996) The    downstream box: an efficient and independent translation initiation    signal in E. coli. EMBO J. 15, 665-674.-   51. Perlman, D. and Halvorson, H. O. (1983) A putative signal    peptidase recognition site and sequence in eukaryotic and    prokaryotic signal peptides. J. Mol. Biol. 167, 391-409.-   52. Von Heijne, G. (1983) Patterns of amino acids near    signal-sequence cleavage sites. Eur. J. Biochem. 133, 17-21.-   53. Pedersen, H., Hölder, S., Sutherlin, D. P., Schwitter, U.,    King, D. S., Schultz, P. G. (1998) Proc. Natl. Acad. Sci. USA. 95,    10523-10528.

1. A method for the selection of a virus comprising the steps of: (a)providing a virus encoding and displaying a fusion polypeptide, saidfusion polypeptide comprising a heterologous polypeptide inserted intothe sequence of a viral coat protein polypeptide, wherein said viruscomprises a cleavable site located within a displayed polypeptide; (b)exposing the virus to a cleaving agent; and (c) propagating the viruscomprising intact fusion protein.
 2. The method according to claim 1,wherein the cleavage site is located within the fusion polypeptide. 3.The method according to claim 2, further comprising separating viruscomprising uncleaved fusion polypeptide from virus comprising cleavedfusion polypeptide.
 4. The method according to claim 1, wherein cleavageimpairs the ability of the polypeptide comprising the cleavage site tomediate the infection of the virus.
 5. The method according to claim 1,wherein the virus encodes a repertoire of sequences.
 6. The methodaccording to claim 5, wherein the repertoire of sequences encodes thedisplayed heterologous peptide or protein.
 7. The method according toclaim 5, wherein the cleavable site is comprised within the repertoireof sequences.
 8. The method according to claim 1, wherein a virus thatis resistant to cleavage is propagated by infection.
 9. The methodaccording to claim 8, wherein the virus which is resistant to cleavagedisplays a folded protein or polypeptide.
 10. The method according toclaim 9, wherein the cleavage is undertaken under conditions at whichsome members of the repertoire are at least partially unfolded.
 11. Themethod of claim 9, wherein the exposing step is undertaken in thepresence of a molecule which stabilises or destabilises the displayedpolypeptide.
 12. The method of claim 11, wherein the exposing step isundertaken in the present of a protein denaturant.
 13. The methodaccording to claim 1, wherein the exposing step is undertaken in thepresence of a ligand for the heterologous polypeptide.
 14. The methodaccording to claim 1, wherein the method persists isolation of a proteinor polypeptide with improved stability.
 15. The method according toclaim 5, wherein the repertoire of sequences encodes a repertoire ofdisplayed proteins which are selected by binding to a ligand.
 16. Themethod according to claim 1, wherein the virus is a bacteriophage. 17.The method according to claim 16, wherein the coat protein is thatprotein encoded by gene 3 of a filamentous bacteriophage.
 18. The methodaccording to claim 17, wherein a cleavage site is introduced between thesecond and third domain of the gene 3 protein.
 19. The method accordingto claim 16, wherein the bacteriophage is a helper bacteriophage used inconjunction with phagemids.
 20. The method according to claim 19,wherein the encapsidated nucleic acid of the bacteriophage is a phagemidand requires the use of a helper bacteriophage.
 21. The method accordingto claim 1, wherein the cleavable site is a protease cleavable site, andthe cleaving agent is a protease.
 22. A method comprising the steps of:(a) creating a library of phagemids encoding a repertoire ofheterologous polypeptides: (b) expressing the phagemids in the presenceof helper phage; (c) subjecting the phage to protease cleavage; (d)infecting bacteria with the phage of step (c); and (e) isolating thephagemids.
 23. A method for effecting helper phage rescue of a phagemidcomprising a fusion polypeptide to form progeny phage, wherein: (a) thehelper phage encodes a viral coat protein comprising aprotease-sensitive cleavage site; (b) the phagemid comprises a fusionpolypeptide; (c) the progeny phage are exposed to a protease capable ofcleaving the protease-cleavable site, such that the cleavage of theprotein derived from the helper phage impairs its ability to mediateinfection, and (d) the progeny are propagated by infection.
 24. Thehelper phage of claim 22, wherein the protease sensitive site inincluded within p3 of the filamentous bacteriophage M13, fd or relatedspecies.
 25. The method according to claim 6, wherein the cleavable siteis comprised within the repertoire of sequences.